Apoptosis denotes the complex contortions of the membrane and organelles of a cell as it undergoes the process of programmed cell death. During said process, the cell activates an intrinsic suicide program and systematically destroys itself in a controlled manner or by a self-regulated process. The following series of events can be observed:                The cell surface begins to bleb and expresses pro-phagocytic signals. The whole apoptotic cell then fragments into membrane-bound vesicles that are rapidly and neatly disposed of by phagocytosis, so that there is minimal damage to the surrounding tissue.        The cell then separates from its neighbors.        The nucleus also goes through a characteristic pattern of morphological changes as it commits genetic suicide. The chromatin condenses and is specifically cleaved to fragments of DNA.        
Neuronal cell death plays an important role in ensuring that the nervous system develops normally. It appears that the death of developing neurons depends on the size of the target that they innervate: cells with fewer synaptic partners are more likely to die than those that have formed multiple synapses. This may reflect a process, which balances the relative number of pre- to postsynaptic neurons in the developing nervous system. Although neuronal cell death was assumed to be apoptotic, it was only recently that neurons in developing rodent brain were conclusively shown to undergo apoptosis as classified by morphology and DNA fragmentation.
Neuronal death occurs via either apoptotic or necrotic processes following traumatic nerve injury or during neurodegenerative diseases. Multiple components are emerging as key players having a role in driving neuronal programmed cell death. Amongst the components leading to neuronal apoptosis are protein members belonging to the Bcl-2 family (see Jacobson, M. D. 1997. Current Biology 7:R 277–R281; Kroemer, G. C. 1997. Nature Medicine: 614–620; Reed, J. C. 1997. Nature 387:773–776).
The entire Bcl-2 family comprises both anti-apoptotic (Bcl-2, Bcl-xL, Bcl-w, Mcl-1, A1, NR-13, BHRF1, LMW5-HL, ORP16, KS-Bcl-2, E1B-19K, CED-9) and pro-apoptotic (Bax, Bak, Bok, Bik, Blk, Hrk, BNIP3, BimL, Bad, Bid, EGL-1) molecules (see Kelekar, A., and C. B. Thomp-son 1998. Trends in Cell Biology 8:324–330). The specific member thereof, i.e. the first found, Bcl-2 is a 26 kDa protein that localizes to the mitochondrial, endoplasmatic reticulum and perinuclear membranes. The Bcl-2 family proteins can form homo- and hetero-dimers that involve amino acid sequences known as Bcl-2 homology (BH) domains. So far, four of said domains (BH1 to 4) have been identified, the BH3 having been attributed a particularly prominent role in view of the death-promoting cascade. Said BH3 domain of the pro-apoptotic members appears to be required for the interaction between anti and pro-apoptotic molecules. The principal site of action of some of the Bcl-2 family members seems to be the mitochondria. Mitochondria have been shown to play a major role in many types of apoptosis. In particular, this organelle has been shown to release Apoptosis Inducing Factor and cytochrome c, a hemoprotein which is bound to the outer surface of the inner mitochondrial membrane. Said cytochrome c has been shown to trigger caspase 9 activation through Apaf-1/caspase 9 complex formation. Bcl-2 family members play a key role in regulating cytochrome c release. While Bcl-2 and Bcl-xL have been shown to suppress cytochrome c release, Bax has been found to stimulate this event both in vitro using isolated mitochondria as well as in intact cells following heterologous expression (Martinou et al.; The Journal of Cell Biology, 128, 1995, 201–208). The mechanisms by which these proteins perform their function are currently unknown. The three-dimensional structure of Bcl-xL and Bid revealed structural similarities between these proteins and the channel-forming domains of the bacterial toxins colicins and diphtheria toxins. Consistent with such structural similarity, some members of this family including Bax were also found able to form ion channels in synthetic lipid membranes.
Studies performed with Bax-deficient mice led to the conclusion that Bax plays a promi-nent role within the apoptosis pathways, notably in neuronal apoptosis. Bax is viewed to be essential for apoptosis induced by NGF deprivation in neonatal sympathetic neurons or for apoptosis induced in cerebellar granule cells by potassium deprivation from the culture medium. Moreover, it was found that in the Bax-deficient mice (knock-out) neonatal moto-neurons from the facial nucleus can survive following axotomy (see Deckwerth, T. L., Elliott J. L., Knudson C. M. et al. 1996. Neuron 17,401–41). Hence, the inhibition of the Bax activity leading to the prevention of cytochrome c release from mitochondria during apoptosis, is viewed to be useful to protect neurons and also other cell types from various cell death stimuli.
In WO 97/01635 (Neurex Corp.) the inhibition of apoptosis in an effort to promote cell survival is suggested to be achieved by introducing into the cell a chimeric gene containing a polynucleotide encoding a Bax-ω-polypeptide (a splice variant of the Bax gene, which displays—in contrast to Bax—an anti-apoptotic activity) being operably linked to a promoter effective to cause transcription of the polynucleotide in the cell. It is reported that the expression of the Bax-ω-polypeptide is effective to inhibit apoptosis in the cell. Perez et al. in Nat. Genet. 1999, 21(2), 200–203 have indicated that apoptosis plays a fundamental role in follicular atresia and they suggest to selectively disrupt the Bax function in order to extend the ovarian lifespan.
Bax down-regulation up to inhibition could indeed represent an interesting therapy for all diseases associated with apoptosis, including neurodegenerative diseases (e.g. Alzheimer's disease, Parkinson's disease, diseases associated with polyglutamine tracts including Huntington's disease, spino-cerebellar ataxias and dentatorubral-pallidoluysian atrophy; amyotrophic lateral sclerosis, retinitis pigmentosa and multiple sclerosis, epilepsy), ischemia (stroke, myocardial infarction and reperfusion injury), infertility (like premature menopause, ovarian failure or follicular atresia), cardiovascular disorders (arteriosclerosis, heart failure and heart transplantation), renal hypoxia, hepatitis and AIDS.
Hence, it is an objective of the present invention to provide compounds enabling the treatment of apoptosis-related disorders, including notably the above mentioned diseases.
It is specifically an objective of the present invention to provide a treatment of apoptosis related disorders by specifically regulating the Bax function, e.g. by modulating, in particular by down-regulating up to inhibiting, the Bax function or by down-regulating, up to inhibiting, the Bax activation.
It is notably an objective of the present invention to provide small molecule pharmaceuticals, more specifically non-protein or non-peptide molecules that avoid essentially all of the drawbacks arising from the use of large peptides or proteins (e.g. restricted bio-availability as well as problems arising from in vivo intolerance thereto), however, which are suitable for the treatment of a number of diseases associated with abnormal apoptosis. It is particularly an objective of the present invention to provide small molecule chemical compounds being suitable Bax modulators (e.g. compounds inhibiting the Bax function or inhibiting the Bax activation) so to be available for a convenient method of treating diseases involving abnormal apoptosis. Moreover, it is an objective of the present invention to provide methods for preparing said small molecule chemical compounds. It is furthermore an objective of the present invention to provide a new category of pharmaceutical formulations for the treatment of a host of diseases. It is finally an objective of the present invention to provide a method of treating diseases that are caused by abnormal apoptosis.